College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea.

Abstract

Germanium biotite (GB) is an aluminosilicate mineral containing 36 ppm germanium. The present study was conducted to better understand the effects of GB on immune responses in a mouse model, and to demonstrate the clearance effects of this mineral against Porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected pigs as an initial step towards the development of a feed supplement that would promote immune activity and help prevent diseases. In the mouse model, dietary supplementation with GB enhanced concanavalin A (ConA)-induced lymphocyte proliferation and increased the percentage of CD3+CD8+ T lymphocytes. In pigs experimentally infected with PRRSV, viral titers in lungs and lymphoid tissues from the GB-fed group were significantly decreased compared to those of the control group 12 days post-infection. Corresponding histopathological analyses demonstrated that GB-fed pigs displayed less severe pathological changes associated with PRRSV infection compared to the control group, indicating that GB promotes PRRSV clearance. These antiviral effects in pigs may be related to the ability of GB to increase CD3+CD8+ T lymphocyte production observed in the mice. Hence, this mineral may be an effective feed supplement for increasing immune activity and preventing disease.

Effects of germanium biotite (GB) on lymphocyte proliferation in mice. Lymphocytes were with or without mitogens (ConA or LPS), and lymphocyte proliferation was measured as OD540 using an MTT assay. Lymphocyte proliferation in response to ConA was significantly enhanced in the GB-fed mice compared to the control group (*p < 0.05). However, proliferation of the unstimulated or LPS-treated lymphocytes did not differ among the groups. Data are presented the mean ± SD (n = 8).

Effect of GB on T lymphocyte subpopulation ratios in mice. The percentage of CD3+CD8+ T lymphocytes in the spleens of GB-fed mice was significantly increased compared to that observed for the control group (***p < 0.001). However, no significant difference in the percentage of spleen CD3+CD4+ T lymphocytes was observed among the groups. Data are presented as the mean ± SD (n = 8).

Effect of GB on rectal temperature in pigs experimentally infected with PRRSV. Mean rectal temperature of the pigs gradually increased up to 14 days post-infection (DPI) after which the temperature declined until the end of the experiment. Rectal temperature of the 0.3% GB-fed group was lower than that of the control group throughout the experimental period. In particular, the difference in rectal temperature between the two groups was statistically significant at 12 and 14 DPI (*p < 0.05). Data are presented as the mean ± SD (0 ~ 12 DPI: n = six in each group, 14~28 DPI: n = three in each group).

Effects of GB on PRRSV clearance in experimentally infected pigs. At 12 DPI, the viral titer of lung (LUN) tissues from the 0.3% GB-fed pigs was significantly decreased compared that for the control group (*p < 0.05). Moreover, viral titers of lymphoid tissues including bronchial lymph node (BLN), tonsil (TON), and thymus (THY) were significantly decreased in the 0.3% GB-fed animals compared to the control group (*p < 0.05). Data are presented as the mean ± SD (n = 3).

Histopathological features of lung and thymus, and microscopic lesion scores for tissue samples from PRRSV-infected pigs. Pigs fed 0.3% GB (B) had milder cases of interstitial pneumonia at 12 DPI (based on lung histopathological features) compared to the control animals (A). Additionally, lymphoid depletion was less severe in the 0.3% GB-fed group, and clearer separation between the cortex and medulla of the thymus (D) compared to the control group (C) was observed. Lung (LUN) microscopic lesion scores for pigs fed 0.3% GB were significantly decrease compared to those for the control group (*p < 0.05). Moreover, microscopic lesion scores for the lymphoid tissues [bronchial lymph node (BLN), tonsil (TON), and thymus (THY)] from the 0.3% GB-fed group were lower than those for the control group although this difference was not significant. Scale bar = 100 µm. Data are presented as the mean ± SD (n = 3).